PROJECT SUMMARY Noonan syndrome (NS; MIM 163950) is an autosomal dominant developmental disorder affecting approximately 1 in every 1,000 to 2,500 live births. NS is characterized by wide phenotypic variability and approximately 80% of patients with clinically diagnosed NS also have congenital cardiovascular abnormalities, including pulmonary valve stenosis, cardiac hypertrophy, or septal defects. Although NS is a genetically heterogeneous disorder, nearly all genes associated with NS encode proteins that are components or regulators of the RAS mitogen-activated protein kinase (MAPK) signaling pathway. And while understanding of the genetic underpinnings of NS has continued to grow over the last decade, approximately 20% of all NS cases remain genetically elusive. Identifying the remaining NS genes is critical for proper patient diagnosis and management, elucidating genotype-phenotype correlations, family screening and development of potential treatments. Recently, whole exome sequencing (WES) and trio-based genomic triangulation performed on a 15-year-old female with a clinical diagnosis of NS and concomitant cardiac hypertrophy (NS+CH) and her unaffected parents elucidated a de novo variant in MRAS- encoded RAS-related protein 3 (MRAS) as the cause of her disease. Biochemical assays demonstrated a significant increase in MRAS activation for the patient-identified mutant MRAS, p.Gly23Val-MRAS, as well as enhanced activation of both RAS/MAPK pathway signaling and downstream gene expression. Subsequent direct sequencing of MRAS in a cohort of 109 unrelated, genetically elusive NS+CH patients revealed an additional, potentially pathogenic mutation, p.Thr68Ile-MRAS, in one patient supporting the hypothesis that MRAS is a novel NS+CH susceptibility gene. In order to further bolster the evidence that mutations in MRAS cause NS+CH, it is necessary to establish pathogenicity of these mutations in multiple in vitro models. To do this, we will take a multimodal approach utilizing traditional in vitro models as well as patient-derived induced pluripotent stem cells (iPSCs). Gene editing of these iPSCs will further demonstrate that mutations in MRAS not only cause disease, but are in fact sufficient to do so.